Xenopus should be at the forefront of systems biology. The unique combination of biochemistry, genomics, embryology and molecular biology that can be easily used in the Xenopus embryo make it ideal for elucidating the complexities of gene regulation. We will use these strengths to explore the regulatory networks underlying endoderm specification. Endoderm is one of first cell types that emerges during embryogenesis. These cells are important for the initial patterning of the vertebrate and are the precursors to many internal organ systems. Xenopus biologists have extensively explored the signaling pathways that define endoderm;including specific signal transduction pathways like Nodal and associated downstream transcription factor cascades. Yet the gene regulatory networks (GRN) of these transcription factors and the transcriptional responses to them are still largely unexplored in any organism. Furthermore the structure and dynamic behavior of these networks within different cell lineages and through developmental time has never been examined in any vertebrate animal or in an in vitro equivalent. The power of the Xenopus embryo combined with advances in sequencing technology - primarily Chromatin Immunoprecipitation followed by Massively Parallel Sequencing (ChIPSeq) - allow the elucidation of targets used by each transcription factor and the underlying gene regulatory networks that these transcription factors define. We hypothesize that identifying the network used to specify endoderm in Xenopus will answer several important and long sought after biological questions, including what motifs - and combinations of motifs - are used by transcription factors to modulate their activity at different stages of embryogenesis and how binding of these transcription factors, alone or in combinations, correlate with expression and resulting function. We further propose to elucidate how these networks change when signaling components are removed, providing an analysis of which elements are needed for particular pathways. This resource will allow the connection of pathways and intense exploration into basic questions of Xenopus biology. PUBLIC HEALTH RELEVANCE: The Xenopus embryo is one of the model systems that can combine biochemistry, genomics, embryology and molecular biology. This makes it ideal for elucidating the complexities of gene regulation. We will use these strengths to explore the regulatory networks underlying endoderm specification. Endoderm is one of first cell types that emerges during embryogenesis. These cells are important for the initial patterning of the vertebrate and are the precursors to many internal organ systems. Xenopus biologists have extensively explored the signaling pathways that define endoderm;including specific signal transduction pathways like Nodal and associated downstream transcription factor cascades. Yet the gene regulatory networks (GRN) of these transcription factors and the transcriptional responses to them are still largely unexplored in any organism. In this grant we will use new technologies to elucidate the underlying GRN specifically for endoderm. This resource will allow the connection of pathways and intense exploration into basic questions of Xenopus biology.